Direct observation of protein-ligand interaction kinetics.

Internal dynamics on the micro- to millisecond time scale have a strong influence on the affinity and specificity with which a protein binds ligands. This time scale is accessible through relaxation dispersion measurements using NMR. By studying the dynamics of a protein with different concentrations of a ligand, one can determine the dynamic effects induced by the ligand. Here we have studied slow internal dynamics of the N-terminal src homology 2 domain of phosphatidylinositide 3-kinase to probe the role of individual residues for the interaction with a tyrosine-phosphorylated binding sequence from polyoma middle T antigen. While slow dynamic motion was restricted to a few residues in the free SH2 and in the SH2 complex, motion was significantly enhanced by adding even small amounts of ligand. Kinetic rates induced by ligand binding varied between 300 and 2000 s(-1). High rates reflected direct interactions with the ligand or rearrangements caused by ligand binding. Large differences in rates were observed for residues adjacent in the primary sequence reflecting their individual roles in ligand interaction. However, rates were similar for residues involved in the same side chain interactions, reflecting concerted motions during ligand binding. For a subset of residues, exchange must involve structural intermediates which play a crucial role in high-affinity ligand binding. This analysis supports a new view of the dynamics of individual sites of a protein during ligand interaction.